Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

Abstract:Gelatin hydrogels can mimic the microenvironments of natural tissues and encapsulate cells homogeneously, which makes them attractive for cartilage tissue engineering. Both the mechanical and biochemical properties of hydrogels can affect the phenotype of chondrocytes. However, the influence of each property on chondrocyte phenotype is unclear due to the difficulty in separating the roles of these properties. In this study, we aimed to study the influence of hydrogel stiffness on chondrocyte phenotype while excluding the role of biochemical factors, such as adhesion site density in the hydrogels. By altering the degree of methacryloyl functionalization, gelatin hydrogels with different stiffnesses of 3.8, 17.1, and 29.9 kPa Young's modulus were prepared from the same concentration of gelatin methacryloyl (GelMA) macromers. Bovine articular chondrocytes were encapsulated in the hydrogels and cultured for 14 days. The influence of hydrogel stiffness on the cell behaviors including cell viability, cell morphology, and maintenance of chondrogenic phenotype was evaluated. GelMA hydrogels with high stiffness (29.9 kPa) showed the best results on maintaining chondrogenic phenotype. These results will be useful for the design and preparation of scaffolds for cartilage tissue engineering.

In contrast to one-photon microscopy, two-photon probe-based fluorescent imaging can provide improved three-dimensional spatial localization and increased imaging depth. Consequently, it has become one of the most attractive techniques for studying biological events in living cells and tissues. However, the quantitation of these probes is primarily based on single-emission intensity change, which tends to be affected by a variety of environmental factors. Ratiometric probes, on the other hand, can eliminate these interferences by the built-in correction of the dual emission bands, resulting in a more favorable system for imaging living cells and tissues. Herein, for the first time, we adopted a through-bond energy transfer (TBET) strategy to design and synthesize a small molecular ratiometric two-photon fluorescent probe for imaging living cells and tissues in real time. Specifically, a two-photon fluorophore (D-π-A-structured naphthalene derivative) and a rhodamine B fluorophore are directly connected by electronically conjugated bond to form a TBET probe, or Np-Rh, which shows a target-modulated ratiometric two-photon fluorescence response with highly efficient energy transfer (93.7%) and two well-resolved emission peaks separated by 100 nm. This novel probe was then applied for two-photon imaging of living cells and tissues and showed high ratiometric imaging resolution and deep-tissue imaging depth of 180 μm, thus demonstrating its practical application in biological systems.

H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S in living systems are desired. Although quite a few one photon fluorescence probes have been reported for H2S, two-photon (TP) probes are more favorable for intracellular imaging. In this work, by employing a donor-π-acceptor-structured naphthalene derivative as the two-photon fluorophore and an azide group as the recognition unit, we reported a new two-photon bioimaging probe 6-(benzo[d]thiazol-2'-yl)-2-azidonaphthalene (NHS1) for H2S with improved sensitivity. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for both one photon and TP excited fluorescence were observed, resulting in a high sensitivity to H2S in aqueous solutions with a detection limit of 20 nM observed, much lower than the previously reported TP probe. The probe also exhibits a wide linear response concentration range (0-5 μM) to H2S with high selectivity. All these features are favorable for direct monitoring of H2S in complex biological samples. It was then applied for direct TP imaging of H2S in living cells with satisfactory sensitivity, demonstrating its value of practical application in biological systems.